The present invention relates to a polymeric composition, which is a crosslinked water swellable polymer, the hydrogel of which is transparent and has a high refractive index, rendering it useful for use in a refractive device, for instance an intraocular lens.
Various products have been developed for replacing or augmenting the natural lens. Replacement lenses may be used where the original lens is clouded by cataracts. Lenses which augment the natural lens and which are intended to be inserted into the eye, include intraocular contact lenses, corneal implants, corneal inlays and corneal onlays.
A method of implanting an intraocular lens in a rolled up form, to minimise the size of the incision is in widespread use. Such a device is described in U.S. Pat. Nos. 4,573,998 and 4,702,244. In these specifications, the material of the lens is described as having a shape memory. The device thus recovers its original conformation after being released from the restraining insertion device through which it is introduced. The above specifications do not describe in any detail the materials used to form the lens.
In U.S. Pat. No. 4,608,049, a foldable intraocular lens is formed of a silicone rubber or a crosslinked hydroxyethylmethacrylate:N-vinyl-2-pyrrolidone:methacrylic acid polymer, that is a water-swellable material.
Further descriptions of hydrogel intraocular lenses are by Barrett in U.S. Pat. No. 4,664,666. Barrett uses a hydrogel of hydroxyethylmethacrylate, a common component of hydrogel contact lens compositions. One problem with poly HEMA hydrogels is that the refractive index of the gel is relatively low. It is preferred for a hydrogel to have higher refractive indices, for instance at least 1.45, up to around 1.60.
Higher refractive index materials are described in EP-A0485197. The polymers must be formed of at least two aryl acrylate polymers, for instance 2-phenylethyl acrylate and 2-phenylethyl methacrylate. The crosslinker is selected from aliphatic diacrylates. The refractive indices of the material is in the range 1.553 to 1.556. The polymers are not, however, hydrogels (that is they are not water-swellable).
EP-A-0308130 describes elastic intraocular lenses formed of copolymers of methacrylate and acrylate esters which are respectively relatively hard and relatively soft at body temperature. The monomers are all aliphatic acrylates. The crosslinker is an aliphatic dimethacrylate.
In our earlier application WO-A-9207885, we describe crosslinked polymers formed of zwitterionic monomer and nonionic copolymerisable monomer, and hydrogel lenses formed therefrom. The examples all used an alkyl acrylate or hydroxyalkylacrylate comonomer. The crosslinking monomers were all aliphatic di-ethylenically unsaturated compounds.
In EP-A-0563299 a copolymer of a zwitterionic monomer and a nonionic monomer is used as a contact lens. In the worked examples, comonomers are hydroxyethylmethacrylate, N-vinylpyrollidone and methylmethacrylate. Crosslinkers are all aliphatic compounds (allyl methacrylate and diethylene glycol di-methacrylate).
In U.S. Pat. Nos. 5,391,669 and 5,270,415, hydrogels formed of balanced charge ion pairs and nonionic comonomer are used as contact lenses. The balanced charge ion pair may be a zwitterionic monomer. In the worked examples, the nonionic comonomers are selected from hydroxyethyl methacrylate, silyl group containing monomers, alkyl methacrylates, hydroxypropyl methacrylate, fluoroalkyl methacrylate and hydroxypropyl methacrylamide. Crosslinkers used in the worked examples are all aliphatic compounds.
A new crosslinked polymer according to the invention is obtainable by radical polymerisation of ethylenically unsaturated monomers including
a) a zwitterionic monomer of the general formula I
YBXxe2x80x83xe2x80x83I
wherein
B is a straight or branched alkylene, oxaalkylene or oligo-oxaalkylene chain optionally containing one or more fluorine atoms up to and including perfluorinated chains or, if X or Y contains a terminal carbon atom bonded to B, a valence bond;
X is a zwitterionic group; and
Y is an ethylenically unsaturated polymerisable group selected from 
CH2xe2x95x90C(R)xe2x80x94CH2xe2x80x94Oxe2x80x94, CH2xe2x95x90C(R)xe2x80x94CH2OC(O)xe2x80x94, CH2xe2x95x90C(R)OC(O)xe2x80x94, CH2xe2x95x90C(R)xe2x80x94Oxe2x80x94, CH2xe2x95x90C(R)CH2OC(O)N(R1)xe2x80x94, R2OOCCRxe2x95x90CRC(O)xe2x80x94Oxe2x80x94, RCHxe2x95x90CHC(O)Oxe2x80x94, RCHxe2x95x90C(COOR2)CH2xe2x80x94C(O)xe2x80x94Oxe2x80x94, 
wherein:
R is hydrogen or a C1-C4 alkyl group;
R1 is hydrogen or a C1-C4 alkyl group or R1 is xe2x80x94Bxe2x80x94X where B and X are as defined above; and
R2 is hydrogen or a C1-4alkyl group or BX where B and X are as defined above;
A is xe2x80x94Oxe2x80x94 or xe2x80x94NR1xe2x80x94;
K is a group xe2x80x94(CH2)pOC(O)xe2x80x94, xe2x80x94(CH2)pC(O)Oxe2x80x94, xe2x80x94(CH2)pOC(O)Oxe2x80x94, xe2x80x94(CH2)pNR3xe2x80x94, xe2x80x94(CH2)pNR3C(O)xe2x80x94, xe2x80x94(CH2)pC(O)NR3xe2x80x94, xe2x80x94(CH2)pNR3C(O)Oxe2x80x94, xe2x80x94(CH2)pOC(O)NR3xe2x80x94, xe2x80x94(CH2)pNR3C(O)NR3xe2x80x94 (in which the groups R3 are the same or different), xe2x80x94(CH2)pOxe2x80x94, xe2x80x94(CH2)pSO3xe2x80x94, or, optionally in combination with B, a valence bond and p is from 1 to 12 and R3 is hydrogen or a C1-C4 4 group.
b) an aromatic group containing monomer of the general formula II
Y1R4xe2x80x83xe2x80x83II
wherein Y1 is selected from 
CH2xe2x95x90C(R5)xe2x80x94CH2xe2x80x94Oxe2x80x94, CH2xe2x95x90C(R5)xe2x80x94CH2OC(O)xe2x80x94, CH2xe2x95x90C(R5)OC(O)xe2x80x94, CH2xe2x95x90C(R5)xe2x80x94Oxe2x80x94, CH2xe2x95x90C(R5)CH2OC(O)N(R6)xe2x80x94, R7OOCCR5xe2x95x90CR5C(O)xe2x80x94Oxe2x80x94, R5CHxe2x95x90CHC(O)Oxe2x80x94, R5CHxe2x95x90C(COOR7)CH2xe2x80x94C(O)xe2x80x94Oxe2x80x94, 
wherein:
R5 is hydrogen or a C1-C4 alkyl group,
R6 is hydrogen or a C1-C4 alkyl group or R5 is R3;
R7 is hydrogen or a C1-4 alkyl group or R3;
A1 is xe2x80x94Oxe2x80x94 or xe2x80x94NR5xe2x80x94; and
K1 is a group xe2x80x94(CH2)qOC(O)xe2x80x94, xe2x80x94(CH2)qC(O)Oxe2x80x94, xe2x80x94(CH2)qOC(O)Oxe2x80x94, xe2x80x94(CH2)qNR8xe2x80x94, xe2x80x94(CH2)qNR8C(O)xe2x80x94, xe2x80x94(CH2)qC(O)NR8xe2x80x94, xe2x80x94(CH2)qNR8C(O)Oxe2x80x94, xe2x80x94(CH2)qOC(O)NR8xe2x80x94, xe2x80x94(CH2)qNR8C(O)NR8xe2x80x94 (in which the groups R8 are the same or different), xe2x80x94CH2)qOxe2x80x94, xe2x80x94(CH2)qSO3xe2x80x94, or a valence bond and p is from 1 to 12 and R8 is hydrogen or a C1-C4 alkyl group;
and R4 is an aromatic group; and
c) a cross-linking monomer of the general formula III
(Y2)nR9xe2x80x83xe2x80x83III
in which n is an integer of at least 2, each Y2 is selected from 
CH2xe2x95x90C(R10)xe2x80x94CH2xe2x80x94Oxe2x80x94, CH2xe2x95x90C(R10)xe2x80x94CH2OC(O)xe2x80x94, CH2xe2x95x90C(R10)OC(O)xe2x80x94, CH2xe2x95x90C(R10)xe2x80x94Oxe2x80x94, CH2xe2x95x90C(R10)CH2OC(O)N(R11)xe2x80x94, R12OOCCR10xe2x95x90CR10C(O)xe2x80x94Oxe2x80x94, R10CHxe2x95x90CHC(O)Oxe2x80x94, R10CHxe2x95x90C(COOR12)CH2xe2x80x94C(O)xe2x80x94Oxe2x80x94, 
wherein:
R10 is hydrogen or a C1-C4 alkyl group;
R11 is hydrogen or a C1-C4 alkyl group or R11 is R3;
R12 is hydrogen or a C1-4 alkyl group or R3;
A2 is xe2x80x94Oxe2x80x94 or xe2x80x94NR11xe2x80x94;
K2 is a group xe2x80x94(CH2)rOC(O)xe2x80x94, xe2x80x94(CH2)rC(O)Oxe2x80x94, xe2x80x94(CH2)rOC(O)Oxe2x80x94, xe2x80x94(CH2)rNR12xe2x80x94, xe2x80x94(CH2)rNR13C(O)xe2x80x94, xe2x80x94(CH2)rC(O)NR13xe2x80x94, xe2x80x94(CH2)rNR13C(O)Oxe2x80x94, xe2x80x94(CH2)rOC(O)NR13xe2x80x94, xe2x80x94(CH2)rNR13C(O)NR13xe2x80x94 (in which the groups R13 are the same or different), xe2x80x94(CH2)rOxe2x80x94, xe2x80x94(CH2)rSO3xe2x80x94 or a valence bond and r is from 1 to 12 and R13 is hydrogen or a C1-C4 alkyl group;
and R9 is an n-functional organic group.
Suitable examples of aromatic groups R4 are optionally substituted aralkyl and alkaryl groups. Most preferably, a group R4 is an unsubstituted aryl or aralkyl group, in which the alkyl group has 1 to 4 carbon atoms, for instance benzyl, 2-phenylethyl or phenyl.
For optimum copolymerisability, the groups Y, Y1 and Y2 have the same general definition. Most preferably each such group is an (alk)acrylic or a styrenic group. An acrylic group, H2Cxe2x95x90C(H or Me)COxe2x80x94(O or NH) are particularly preferred. Preferably all such groups are either methacrylic R=R5=R10=Me) or acrylic (R, R5, R10=hydrogen), and are preferably all ester or amide derivatives thereof. Most conveniently, the monomers are all acrylic esters, generally either methacrylate esters or acrylate esters.
In the invention, it is found that optimum crosslinking of the aromatic and zwitterionic monomers is achieved where a crosslinking monomer of the formula III in which the group R9 is an aromatic group is included. Suitable aromatic groups are, for instance, phenylene, alkarylene, aralkylene, and bisphenol A-type groups. Most preferably the crosslinker includes bisphenol A dimethacrylate. The crosslinker may be di-, tri-, tetra- or higher functional, for instance an oligomeric or polymeric compound.
It is found to be particularly preferred for the crosslinking monomer to include a monomer of the general formula III in which the group R9 is an aliphatic group. Suitable aliphatic groups R9 are C2-8-alkylene, C2-4-alkyleneoxy-C2-4-alkylene or oligo(C2-4-alkyleneoxy)-C2-4-alkylene (e.g. xe2x80x94(CH2CH2O)tCH2CH2xe2x80x94, where t is 1-50).
Most preferably a mixture of crosslinking agents is included, including at least one crosslinking agent in which R9 is an aromatic group and at least one crosslinking agent in which R9 is an aliphatic group.
In the general formula I, the zwitterionic group preferably has the general formula IV 
in which the moieties X4 and X5, which are the same or different, are xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94 or a valence bond, preferably xe2x80x94Oxe2x80x94, and W+ is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C2-12 alkylene group,
preferably in which W+ is a group of formula xe2x80x94W1xe2x80x94N+R143, xe2x80x94W1xe2x80x94P+R153, W1xe2x80x94S+R152 or xe2x80x94W1-Het+ in which:
W1 is alkylene of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W1 optionally contains one or more fluorine substituents and/or one or more functional groups; and
either the groups R14 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl or two of the groups R14 together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R14 together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R14 is substituted by a hydrophilic functional group, and
the groups R15 are the same or different and each is R14 or a group OR14, where R14 is as defined above; or
Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.
Most preferably, the zwitterionic group of the formula IV, has the general formula V: 
where the groups R16 are the same or different and each is hydrogen or C1-4 alkyl, and m is from 1 to 4, in which preferably the groups R16 are the same.
Alternatively, the zwitterionic group may be a betaine group (ie in which the cation is closer to the backbone), for instance a sulpho-, carboxy- or phospho-betaine. A betaine group should have no overall charge and is preferably therefore a carboxy- or sulpho-betaine. If it is a phosphobetaine the phosphate terminal group must be a diester, i.e., be esterified with an alcohol. Such groups may be represented by the general formula VI
xe2x80x94X2xe2x80x94R17xe2x80x94N⊕(R18)2xe2x80x94R19xe2x80x94Vxe2x8ax96xe2x80x83xe2x80x83VI
in which X2 is a valence bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94NHxe2x80x94, preferably xe2x80x94Oxe2x80x94;
V is a carboxylate, sulphonate or phosphate diester(monovalently charged) anion;
R17 is a valence bond (together with X2) or alkylene xe2x80x94C(O)alkylene- or xe2x80x94C(O)NHalkylene preferably alkylene and preferably containing from 1 to 6 carbon atoms in the alkylene chain;
the groups R18 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms or the groups R18 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 atoms; and
R19 is alkylene of 1 to 20, preferably 1 to 10, more preferably 1 to 6 carbon atoms.
One preferred sulphobetaine monomer has the formula VII 
where the groups R20 are the same or different and each is hydrogen or C1-4 alkyl and s is from 2 to 4.
Preferably the groups R20 are the same. It is also preferable that at least one of the groups R20 is methyl, and more preferable that the groups R20 are both methyl.
Preferably s is 2 or 3, more preferably 3.
Alternatively the zwitterionic group may be an amino acid moiety in which the alpha carbon atom (to which an amine group and the carboxylic acid group are attached) is joined through a linker group to the backbone of polymer A. Such groups may be represented by the general formula VIII 
in which X3 is a valence bond, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94 or xe2x80x94NHxe2x80x94, preferably xe2x80x94Oxe2x80x94,
R21 is a valence bond (optionally together with X3) or alkylene, xe2x80x94C(O)alkylene- or xe2x80x94C(O)NHalkylene, preferably alkylene and preferably containing from 1 to 6 carbon atoms; and
the groups R22 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or two of the groups R19, together with the nitrogen to which they are attached, form a heterocyclic ring of from 5 to 7 atoms, or the three group R22 together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring.
The mole ratio of zwitterionic monomer to aromatic group containing monomer is generally in the range 1:99 to 99:1, preferably 1:20 to 1:1, more preferably in the range 1:10 to 1:2. The amount of zwitterionic monomer in total monomer is preferably in the range 1 to 95%, more preferably 5 to 50%, most preferably 10 to 25%. The amount of aromatic group containing monomer is preferably in the range 10 to 99%, more preferably 50 to 95%, most preferably 75 to 90%.
In the polymerisation mixture, the crosslinking monomer is generally present in a molar amount in the range 0.01% to 10%, most preferably in the range 0.1 to 5% based on total moles of monomer. Where a mixture of aromatic group containing cross-linking monomer to aliphatic group containing monomer is used the molar ratio of the two is preferably in the range 10:1 to 1:10, preferably 5:1 to 1:5, more preferably 2:1 to 1:2 most preferably 3:2 to 2:3.
The zwitterionic group containing monomer is generally included in sufficient levels to render the polymer swellable in water and to render the hydrogel more biocompatible.
According to a further aspect of the invention, there is provided a hydrogel formed of the novel crosslinked polymer and, dispersed throughout the polymer, an aqueous liquid. The water content of the polymer when fully swollen in deionised water is preferably in the range 10 to 50%, for instance in the range 20 to 40%, most preferably in the range 25 to 35%.
Preferably the hydrogel (the polymer swollen in water) is transparent. It is found that the hydrogel of the invention has a high transmission rate for visible light. The average transmission rate should preferably be above 90% throughout the range of visible light, 400 to 700 nm wavelengths.
The incorporation of the aromatic monomer enables high refractive indices to be achieved. Thus the refractive index of the fully water swollen hydrogel, may be at least 1.45, for instance up to 1.60. Preferably the refractive index is in the range 1.45 to 1.55.
The present invention includes also a polymerisation process, in which the mixture of monomers a, b and c are subjected to conditions whereby polymerisation is initiated and propagated. Initiation may be by any suitable means, for instance using thermal, redox or UV initiators, optionally in combination with one another. The polymer of the invention, when used as an intraocular lens, may include an absorber of ultraviolet light.
Since the zwitterionic monomer tends to be very polar and aromatic monomers tend to be non polar, the monomers may be immiscible with one another. In order to achieve a homogenous polymerisation mixture therefore it may be necessary to include a non polymerisable diluent liquid which acts as a common solvent for the monomers. A suitable solvent is an alcohol. The solvent is generally removed from the product polymer after polymerisation, for instance by evaporation or by solvent replacement using an alternative liquid, generally water or other aqueous solution.
It is generally necessary to include any non polymerisable liquid diluent in an amount in the range 5 to 90% by weight based on the total weight of the polymerisation mixture. In order to avoid unnecessary solvent removal, the level is preferably less than 75%, for instance less than 50%. It is generally necessary to include at least 10% to achieve adequate dissolution of the monomers.
The polymerisation is generally conducted in some form of mould, for instance to form precursor products from which shaped lenses may be formed. Where such products are for instance rods, buttons or other lens precursors, shaping to form appropriate three dimensional shapes is generally by lathing. In order for lathing to take place, it is generally necessary to remove any polymerization diluent prior to carrying out the lathing step.
Alternatively the lens or other final product may be polymerised in a mould of the desired final shape. In this case, the solvent is removed after polymerisation, for instance by solvent replacement.
For any polymerisation method, a final step in the formation of a hydrogel product involves swelling the crosslinked polymer in an aqueous liquid. The materials, when swollen in water have very desirable mechanical properties, for instance enabling them to be used as foldable IOL""s. The strain at break for the swollen materials may be at least 50%, or even more than 100%. The modulus should preferably be in the range 1 to 4 MPa.
The materials (xerogels) have hardness values high enough to render them suitable for shaping by machining, for instance, by lathe cutting.
The product is found to have very desirable mechanical, optical and biocompatible properties rendering it suitable for use as a refractive device. The polymers are of particular value for use as intraocular devices especially intraocular lenses (IOL""s) such as replacement lenses, lenses to augment the natural lens, e.g. posteria chamber phakic IOL""s, anterior chamber phakic IOL""s, corneal implants such as corneal inlays, corneal onlays and intracorneal rings.
The improved biocompatibility resulting from the incorporation of a zwitterionic monomer is believed to result in less damage being caused by a phakic lens on the natural lens (avoiding cataract formation) or on the iris by chafing. For any intraocular device the improved biocompatibility should result in reduced inflammatory response. The results presented hereinafter show that the materials cause less endothelial damage than prior art materials.
The following examples illustrate the invention.
Abbreviations
EWC=Equilibrium Water Contact
RI=Refractive Index
Trans=Optical transmission
BA=Benzyl acrylate
HEMA-PC=2-Methacryloyloxyethyl-2xe2x80x2-trimethylammonium ethyl phosphate inner salt
LM=Lauryl Methacrylate
EGDMA=Ethylene glycol dimethacrylate
BADMA=Bisphenol-A dimethacrylate
FEM=Fluoroethyl Methacrylate
AIBN=Azoisobutyronitrile
General Polymerisation Method
The monomers, including the crosslinking monomer, in the desired quantities, were dissolved in dry ethanol, in an amount of 20% (based on total polymerization mixture weight) unless otherwise specified. Initiator of the desired type and in the desired amount was dissolved into the mixture (AIBN, unless otherwise specified). The liquid polymerization mixture was thoroughly degassed using nitrogen. The polymerisation mixture was then injected into the desired mould (to form a membrane or button, as the case may be) which bad previously been flushed with nitrogen. The mould was subsequently sealed and suspended in a water bath (containing oxygen scavenger) at the desired temperature (usually 60xc2x0 C.) for the desired period (usually sixteen hours) to complete polymerisation. Unless otherwise specified, the polymer, still in the mould after removal from the water bath, was annealed under vacuum at 90xc2x0 C. for a further sixteen hours, before being removed from the moulds. For all button polymerisations, the buttons, after removal from the mould, were annealed at 110xc2x0 C. under vacuum for 4 days.
Mechanical Properties
EWC
The EWC is measured by weighing hydrogel lenses in their fully hydrated state and after drying in an oven overnight at 110xc2x0 C.
Refractive Index
The retractive index is measured on an Atago device with the material in fully swollen (in water) form.
Hardness
The hardness of the F-type button (from which a lens is cut) is measured using a Type D Shore Durometer. A cross section is cut from the button using a microslicer or lathe and the centre and edge hardness recorded. This test is carried out on the xerogel.
Expansion Factor
This is determined from the ratio of diameter of lens hydrated to diameter of lens dry.
Tensile Properties
The mechanical analysis is carried out as a tensile test using a Mini Instron 44, using a 5 load cell at a speed of 5 mm per minute. The material is tested at 25+/xe2x88x922xc2x0 C. and kept hydrated throughout the test.
Biological Properties
The following tests are carried out on disks cut from polymer membranes, unless otherwise specified.
Fibrinogen Adhesion
This assay quantifies protein deposited on a test surface using polycolonal anti serum and enzyme-conjugated secondary anti serum. It provides a useful assessment of haemocompatibility and general biocompatibility.
Buttons of test material are each placed into a well of a twenty four well plate.
One microgram fibrinogen in 50 xcexcl PBS. The fibrinogen solution is left in contact with the samples for two hours at room temperature. Subsequently the samples are washed three times with PBS, excess binding sites are blocked by overnight incubation with 4% bovine serum albumin in PBS at 4xc2x0 C. The buttons are subsequently washed three times with PBS and replaced into fresh wells of a new 24 well plate. 500 xcexcl diluted antithuman fibrinogen antiserum (1:5000 in PBS) is added and incubated with the samples for 30 minutes at room temperature. The buttons are washed three times in PBS and placed into fresh wells of a 24 well plate. 500 xcexcl diluted horseradish peroxidase-conjugated rabbit anti-goat antiserum (1:500 in PBS) is added and incubated for 30 minutes at room temperature. The buttons are washed three times in PBS and placed into fresh wells of a further 24 well plate. A substrate for the peroxidase enzyme is subsequently added in an appropriate buffer after contact with the samples for 10 minutes at room temperature. A terminator is added and the solutions read in a suitable colour remitter. The results are reported in terms of relative light units as compared to PMMA and pHEMA controls. High values thus correspond to high levels of adhesion.
Bacterial Adhesion Assay
This assay measures the bacterial (Staphylococcus epidermidis) attachment on the test materials using extraction of cellular ATP of attached bacterial cells. Extracted ATP is evaluated using bioluminescent technique. If bacteria adhere to and are carried with an intraocular lens into the eye, they could cause an infection.
Samples of the material under test are placed in the desired number of wells in a microtitre plate. A bacterial suspension at 3xc3x97108 CFU per ml in phosphate buffered saline is incubated in the wells for four hours at 37xc2x0 C. under agitation. The bacterial suspension is aspirated from the wells and samples subsequently rinsed in sterile phosphate buffered saline before being placed into the wells of a new plate. Lysis buffer (0.1% trichloroacetic acid, 1% xylenol 2 mM EDTA in deionised water) is placed into the well and left in contact with the sample for 10 minutes to extract the ATP. Extracted ATP is diluted with tris acetate buffer 1:1, ATP monitoring reagent is added and light emission determined in a bioluminescent plate reader. Bioluminessence is related to a number of bacterial cells by generating a calibration curve.
Fibroblast Adhesion Assay
This assay determines the adhesion of a standard adherent cell line, mouse 3T3 fibreblasts, in tissue culture medium, to samples on the test.
The fibroblast cells are cultivated in a tissue culture step to confluent or near confluent monolayer. The monolayer is detached from the flask using a solution of trypsin-EDTA, and subsequently suspended in serum-containing medium. Test disks of the material under test of 13 mm diameter are placed into twenty four well plates. 0.5 mls of cell suspension (having a cell concentration of 3000 per ml) is added and incubated for 72 hours at 37xc2x0 C. The samples are removed from the wells and washed with PBS. The samples with adherent cells are placed into wells of a new plate and subjected to lysis for 30 minutes at room temperature followed by freezing overnight. After addition of further lysis buffer, ATP monitoring reagent is added to the wells and luminescence subsequently read to determine ATP levels. The results are reported in terms of relative light units as compared to standard materials (polymethylmethacrylate and polyhydroxyethylmethacrylate).
Granulocyte Activation
This method measures granulocyte activation in response to superoxide radicals by disks of materials under test. It is a useful measure of biocompatibility. The cells subjected to the test are polymorphonuclear leucocytes (PMN""s) from venus blood. Incubation of the cells in the presence of the materials under test is conducted in the presence of nitroblue tetrazolium, which detects the oxidative burst triggered by inflammatory materials upon granulocyte activation to be visualised colorimetrically.
PMN""s are separated from venus blood using a suitable technique. They are suspended in Earl""s salt solution containing 10% foetal calf serum at a cell density of 1xc3x97106 cells per ml. 100 xcexcl of the cell suspension is contacted with 13 mm disks of samples under test in the wells of a microtitre plate. After 30 minutes at 37xc2x0 C., the samples in the wells are washed three times with phosphate buffered saline and then 100 xcexcl of nitroblue tetrazolium is added to the wells. Adherent cells are incubated with the NBT solution for at least one hour at 37xc2x0 C. Subsequently cells are fixed using formaldehyde, washed and viewed under light microscopy. Activated granulocytes appear blue. The number of activated granulocytes as compared to polymethylmethacrylate controls and positive controls (polymethylmethacrylate treated with phorbol ester as a positive control).
Macrophage Adhesion
This protocol measures macrophage adhesion to test surfaces, another useful measure of biocompatibility. The protocol involves incubation with novel materials overnight which exploits the known tendency of macrophages to attach to plastics surfaces.
Suitable purification steps are conducted to separate out mononuclear cells from venus blood. The cells are suspended in serum-free macrophage medium (commercially available) at a concentration of 106 cells per ml. 200 xcexcl of medium is added to wells of a microtitre plate containing 13 mm diameter disks of materials under test. The plates are left overnight at 37xc2x0 C. after which disks are moved to a new plate and washed three times before fixing with formaldehyde and staining with Dako anti-macrophage antibody-biotin conjugate. Attached antibody is subsequently visualised using a Sigma high intensity rapid stain kit containing an avidin-peroxidase conjugate with 3-amino-9-ethyl carbazole (AEC) as chromogen. The numbers of macrophages is determined using light microscopy and compared against controls.
Rabbit Epithelial Lens Cell Adhesion
AGO4677, a mortal primary rabbit lens epithelial cell strain was obtained from the National Institute of Aging (NIA) repository (Cambden, N.J., USA). The cells were cultures in Minimal Essential Eagles Medium (MEM) with double the normal concentration of non-essential amino acids (Gibco) and 10% (v/v) foetal calf serum (FCS) at a density of 6000 cells cmxe2x88x922 at 37xc2x0 C. in 5% CO2. The cells were never permitted to become confluent under maintenance conditions and were passaged by routine trypsin dispersion. Adhesion of the AGO4677 was assayed by ATP extraction 72 h after 1000 viable rabbit lens cells were plated onto discs of material. ATP was liberated by incubating each disk with 100 xcexcl of a sterile hypotonic lysis buffer (0.01M Tris-Acetate pH 8, 2 mM EDTA). The ATP solution was then diluted 1:1 with a commercial assay buffer designed for ATP luminometry (0.1 M Tris-Acetate pH 8, 2 mM EDTA) and the amount of ATP in each sample measured using a commercial kit (BioOrbit-Wallac, Turku Finland) and a 96 well plate luminometer (Amerlite, Amersham).
Corneal Endothelial Cell Touch Test
This test method is a highly discriminating assay for intraocular lens utility. The test involves contacting materials under test with a confluent monolayer of bovine corneal endothelium (BCE) cells for a predetermined period of time and qualitatively assessing the damage to the monolayer.
Confluent monolayers of BCE cells in sterile tissue culture dishes are established by culturing in the presence of dulbecco""s modified eagles medium (DMEM) containing foetal bovine serum, new born calf serum, penicillin and streptomycin. The monolayers were rinsed to remove unattached cells. A sterile 13 mm disk of test sample (xerogel) is rinsed ten times with sterile PBS to hydrate. The hydrated disk is then placed on the BCE monolayer surface and a weight placed upon the top of a disk to ensure contact (in the xe2x80x9cAv Damagexe2x80x9d, and HamaIP tests (see Table 7) a 2 g weight was used, whilst a lighter weight was used for the xe2x80x9ccell damagexe2x80x9d results). After a predetermined period of time (3xc2xd minutes) the disk is removed, fresh medium is added and the cells incubated for 15 minutes to allow recovery. The cells are then viewed under inverted light microscope and the damage is qualitatively assessed on a numerical scale of 0 (complete destruction) to 10 (minimal damage). Values are given as Av(erage) Damage in results. The cells are also analysed by fixing, staining with Harris hematoxylin solution and viewed using Argus 50 software and a Hamamatsu image processor (Hama IP). The results of the quantitative determination are expressed in terms of mean percent damage (sample number six).